Abstract:

One aspect of the present invention is directed to search receptors based
on the information of HN signaling pathways in order to find Humanin
receptor or Humanin-like polypeptide receptor (HNR), and to reveal a
mechanism of promoting or suppressing the intracellular signal
transduction for neuroprotecting activity of HN and identify a compound
involved in the mechanism. The aspect of the invention is directed to a
method for screening of HNR agonist and HNR antagonist, to utilize the
screened compound in development of a drug for the treatment of
neurodegenerative disease, and to provide an assay system of AD neuronal
cell death, and to provide methods for the compulsory expression of HNR
gene or knocking-out of intracellular genes.

Claims:

1. A Humanin receptor or Humanin-like polypeptide receptor (HNR)
comprising at least two kinds of proteins selected from the group
consisting of gp130 or its partial polypeptide, CNTF receptor a chain
(CNTF-R) and WSX-1.

2. The Humanin receptor or Humanin-like polypeptide receptor (HNR) of
claim 1 consisting of gp130 or its partial polypeptide, CNTF-R and WSX-1.

3. The Humanin receptor or Humanin-like polypeptide receptor (HNR) of
claim 1 consisting of gp130 or its partial polypeptide and WSX-1.

6. The Humanin receptor or Humanin-like polypeptide receptor (HNR) of
claim 1, wherein gp130, CNTF-R and WSX-1 are a protein derived from
human.

7. A method for screening of a compound that binds to the Humanin receptor
or Humanin-like polypeptide receptor (HNR) of claim 1.

8. The method of claim 7, wherein the compound binds to an extracellular
or an intracellular domain of the receptor.

9. (canceled)

10. The method of claim 7, wherein the compound is an agonist or
antagonist for the receptor.

11. (canceled)

12. The method of claim 7, comprising the steps:(a) a step of placing a
subject sample in contact with the Humanin receptor or Humanin-like
polypeptide receptor (HNR) of claim 1 or at least one protein that
constitutes it;(b) a step of determining a binding characteristic between
the receptor and the compound comprised in the subject sample; and(c) a
step of selecting the compound that binds to the receptor.

13. The method of claim 12, wherein a subject sample is placed in contact
with Humanin receptor or Humanin-like polypeptide receptor (HNR) or at
least one protein that constitutes it in the presence of Humanin or
Humanin-like polypeptide.

14. The method of claim 12, wherein the Humanin receptor or Humanin-like
polypeptide receptor (HNR) or at least one protein that constitutes it is
compulsorily expressed in a cell.

15. The method of claim 12, wherein the Humanin receptor or Humanin-like
polypeptide receptor (HNR) is compulsorily expressed by a cell
transformed with an expression vector comprising a gene encoding at least
one protein that constitutes the receptor.

16. The method of claim 12, wherein the binding characteristic between the
receptor and the compound is determined by detecting a change in a
suppressing or inhibiting function for neuronal cell death, or by
detecting increase or decrease of phosphorylation of tyrosine 706 of
STAT3.

17. (canceled)

18. The method of claim 7, which is performed in a cell-free system.

19. A cell transformed with an expression vector comprising a gene
encoding at least one protein that constitutes Humanin receptor or
Humanin-like polypeptide receptor (HNR), which is selected from the group
consisting of gp130, CNTF-R and WSX-1.

21. A cell in which a gene encoding at least one protein that constitutes
Humanin receptor or Humanin-like polypeptide receptor (HNR), which is
selected from the group consisting of gp130, CNTF-R and WSX-1, is knocked
out.

22. The cell of claim 21, which is an ES cell.

23. A knockout animal except human, which is derived from the cell of
claim 22.

24. The knockout animal of claim 23, which is homozygous.

25. The knockout animal of claim 23, which is a rodent.

26. A pharmaceutical composition as an inhibitor of neuronal cell death,
comprising the compound that can bind to the Humanin receptor or
Humanin-like polypeptide receptor (HNR) of claim 1 as an effective
component.

27. A pharmaceutical composition for use of prevention or treatment of
neurodegenerative diseases, Alzheimer's disease, amyotrophic lateral
sclerosis, mad cow disease, or vascular dementia, comprising the compound
that binds to the Humanin receptor or Humanin-like polypeptide receptor
(HNR) of claim 1 as an effective component.

28.-31. (canceled)

32. An antibody that specifically binds to the Humanin receptor or
Humanin-like polypeptide receptor (HNR) of claim 1.

Description:

FIELD

[0001]The invention relates to a Humanin receptor or Humanin-like
polypeptide receptor (both of which may be also referred to hereinafter
as "HNR"), to a transformant cell compulsorily expressing the receptor,
to a method for screening a compound that binds to the receptor, to a
pharmaceutical composition comprising the compounds and the like.

BACKGROUND

[0002]Neuronal loss, which has been considered to be directly linked to
the major neurological manifestations of Alzheimer's disease (AD), is an
important target for AD therapy although the pathological mechanism
leading to neuronal loss still remains unknown. In vitro, a variety of
AD-related insults, such as overexpression of FAD-related mutants and
increased levels of toxic amyloid b peptides (Aβs) derived from
Amyloid-β precursor protein (APP), induce neuronal cell death via
multiple death pathways.

[0006]We have shown that HN is secreted from cells and inhibits neuronal
cell death by AD-related insults from outside of cells via its putative
receptor on the membrane (Hashimoto et al., 2001a; Nishimoto et al.,
2004).

[0007]Most recently, Ying et al. (2004) have reported that HN inhibits
Aβ (1-42)-induced neurotoxicity by binding to pertussis toxin
(PTX)-sensitive G protein-coupled human formylpeptide receptor-like-1
(FPRL-1) as a HN receptor using PC12 neuroblastoma cells. They suggested
that HN blocks Aβ-induced neurotoxicity by competing with Aβ
for FPRL-1. However, after we have studied how FPRL-1 is involved in
HN-mediated protection against AD-related neuronal insults, we found that
FPRL-1 is not involved in HN-mediated neuroprotection in F11 neurohybrid
cells or primary cortical neurons (Hashimoto et al., 2005), indicating
that there are other receptors than FPRL-1, which may mediate HN-induced
neuroprotection.

[0008]In addition, we revealed that STAT3 as well as a certain kind of
tyrosine kinase are involved in HN-mediated neuroprotection (Hashimoto et
al. 2005), suggesting that some cytokine receptor-like receptors are
involved in their signaling pathway.

[0009]gp130 is a component of cytokine receptor common to interleukin-6
(IL-6) receptor family members. gp130-containing receptors are stimulated
by several type I cytokines consisting of IL-6, IL-11,
Leukemia-inhibitory factor (LIF), ciliary neurotrophic factor (CNTF),
OncostatinM (OSM), and Cardiotropin-1. Binding of these cytokines to the
above cognate receptors leads to homodimerization of gp130, or to
heterodimerization between gp130 and a gp130-related receptor such as the
LIF receptor, the OSM receptor or WSX-1 (IL-27 receptor), eventually
transmitting cytokine signals to intracellular signal cascades mediated
by both JAK/STAT and RAS/MAPK signaling pathways (Taga et al., 1997;
Boulay et al., 2003; Boulanger et al., 2004). Most recently, it has been
shown that IL-27 (IL-27p28/EBV-induced gene 3), which belongs to
IL-6/IL-12 family cytokines, modifies Th-1 and Th-2 immunological
response (Yoshida et al., 2004) by binding to WSX-1/gp130 (Plan et al,
2004). CNTF receptor alpha chain (CNTR-R) is a gp130-related receptor,
which does not have an intracellular signaling domain. WO01/021787 and
WO03/097687.

Problems To Be Solved

[0010]The purpose of this invention is therefore to search receptors based
on the information about HN signaling pathway in order to finally find
Humanin receptor or Humanin-like polypeptide receptor (HNR), and to
reveal a mechanism of promoting or suppressing the intracellular signal
transduction for showing neuroprotecting activity of HN and identify a
compound involved in the mechanism, to establish a method for screening
of HNR agonist and HNR antagonist, to utilize the screened compound in
development of a drug for the treatment of neurodegenerative disease,
especially AD, to provide an assay system of AD neuronal cell death, and
to provide methods for the compulsory expression of HNR gene or
knocking-out of intracellular genes.

SUMMARY

[0011]The present invention relates to the following aspects.

[0012]A Humanin receptor or Humanin-like polypeptide receptor (HNR)
comprising at least two kinds of proteins selected from the group
consisting of gp130 or its partial polypeptide, CNTF receptor a chain
(CNTF-R) and WSX-1.

[0018]A method for screening of a compound that binds to Humanin receptor
or Humanin-like polypeptide receptor (HNR) of Claim 1.

[0019]A screening method of Claim 7, wherein the compound that binds to
Humanin receptor or Humanin-like polypeptide receptor (HNR) binds to an
extracellular domain of the receptor.

[0020]A screening method of Claim 7, wherein the compound that binds to
Humanin receptor or Humanin-like polypeptide receptor (HNR) is an agonist
for the receptor.

[0021]A screening method of Claim 7, comprising the steps: [0022](a) a
step of placing a subject sample in contact with Humanin receptor or
Humanin-like polypeptide receptor (HNR) or at least one protein that
constitutes it; [0023](b) a step of determining a binding characteristics
between the receptor and the compound comprised in the subject sample;
and [0024](c) a step of selecting the compound that binds to the
receptor.

[0025]A screening method of Claim 12, wherein a subject sample is placed
in contact with Humanin receptor or Humanin-like polypeptide receptor
(HNR) or at least one protein that constitutes it in the presence of
Humanin or Humanin-like polypeptide.

[0026]A screening method of Claim 12, wherein Humanin receptor or
Humanin-like polypeptide receptor (HNR) or at least one protein that
constitutes it is compulsorily expressed in a cell.

[0027]A screening method of Claim 12, wherein Humanin receptor or
Humanin-like polypeptide receptor (HNR) is compulsorily expressed by a
cell transformed with an expression vector comprising a gene encoding at
least one protein that constitutes the receptor.

[0028]A screening method of Claim 12, wherein the binding characteristics
between the receptor and the compound is determined by detecting a change
in a suppressing or inhibiting function for neuronal cell death.

[0029]A screening method of Claim 12, wherein the binding characteristics
between the receptor and the compound is determined by detecting increase
or decrease of phosphorylation of tyrosine 706 of STAT3.

[0030]A screening method of Claim 7, which is performed in a cell-free
system.

[0031]A cell transformed with an expression vector comprising a gene
encoding at least one protein that constitutes Humanin receptor or
Humanin-like polypeptide receptor (HNR), which is selected from the group
consisting of gp130, CNTF-R and WSX-1.

[0033]A cell in which a gene encoding at least one protein that
constitutes Humanin receptor or Humanin-like polypeptide receptor (HNR),
which is selected from the group consisting of gp130, CNTF-R and WSX-1,
is knocked out.

[0034]A cell of Claim 21, which is an ES cell.

[0035]A knockout animal except human, which is derived from the cell of
Claim 22.

[0036]A knockout animal of Claim 23, which is homozygous.

[0037]A knockout animal of Claim 23, which is a rodent.

[0038]A pharmaceutical composition as an inhibitor of neuronal cell death,
comprising the compound that can bind to Humanin receptor or Humanin-like
polypeptide receptor (HNR) of Claim 1 as an effective component.

[0039]A pharmaceutical composition for use of prevention or treatment of
neurodegenerative diseases, comprising the compound that binds to Humanin
receptor or Humanin-like polypeptide receptor (HNR) of Claim 1 as an
effective component.

[0040]A pharmaceutical composition for use of prevention or treatment of
Alzheimer's disease, comprising the compound that binds to Humanin
receptor or Humanin-like polypeptide receptor (HNR) of Claim 1 as an
effective component.

[0041]A pharmaceutical composition for use of prevention or treatment of
amyotrophic lateral sclerosis, comprising the compound that binds to
Humanin receptor or Humanin-like polypeptide receptor (HNR) of Claim 1 as
an effective component.

[0042]A pharmaceutical composition for use of prevention or treatment of
mad cow disease, comprising the compound that binds to Humanin receptor
or Humanin-like polypeptide receptor (HNR) of Claim 1 as an effective
component.

[0043]A pharmaceutical composition for use of prevention or treatment of
vascular dementia, comprising the compound that binds to Humanin receptor
or Humanin-like polypeptide receptor (HNR) of Claim 1 as an effective
component.

[0045]The present invention has revealed the structure of Humanin receptor
or Humanin-like polypeptide receptor (HNR), and made it possible to
provide a method for screening a compound that can bind to the above
receptor and a pharmaceutical composition comprising said compound and
the like.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046]FIGS. 1 (A)-(D)

[0047]A) F11 cells were transfected with 0.5 μg of the pcDNA3 vector or
pcDNA3-V642I-APP in association with 0.5 μg of the pCAG vector or
pCAG-wild-type mouse gp130 (mgp130wt) or pCAG-mouse gp130 extracellular
domain (mgp130tr). Cell viability was determined by WST-8 assay at 72 hr
after the transfection. Below are shown the results of the expression of
V642I-APP, and mgp130wt or mgp130tr, which were confirmed by immunoblot
analysis (photos).

[0048]B) PCNs were incubated with 25 μM Aβ (upper) in the presence
of 10 μg of recombinant soluble human gp130 (lower "+") or BSA (lower
"-"). Cell viability was determined by WST-8 assay (upper) or Calcein
fluorescence assay (lower) at 72 hr after the onset of treatment with
Aβ.

[0049]C) PCNs were incubated with 25 μM Aβ (1-43) in the presence
of 1 μg of neutralizing anti-mouse gp130 antibody (RX435) or control
IgG. Cell viability and cell mortality were determined by Calcein
fluorescence (left panel) and LDH assays (right panel), respectively, at
72 hr after the onset of treatment with Aβ (1-43).

[0050]D) F11 cells were transfected with 0.5 μg of the pcDNA3 vector or
pcDNA3-V642I-APP in association with 0.5 μg of the pEFBos vector or
pEFBos-wild-type human gp130 (gp130wt). At 24 hr after transfection, the
cells were added with 1 μg of neutralizing anti-mouse gp130 antibody.
Cell mortality (left panel) and cell viability (right panel) were
determined by Trypan blue exclusion assay and WST-8 assay at 72 hr after
transfection. Below are shown the results of the expression of V642I-APP,
and human gp130wt, which were confirmed by immunoblot analysis (photos).

[0051]FIGS. 2 (A)-(E)

[0052]A) F11 cells were transfected with 0.5 μg of the pcDNA3 vector or
pcDNA3-APP in association with 0.5 μg of the pEFBos vector or the
pEFBos vector encoding the extracellular domain of the human G-CSF
receptor fused to the full transmembrane domain and a C-terminally
truncated intracytoplasmic (intracellular) domain of gp130 (named
"G-CSFR/gp130"). The results of the expression of each chimeric proteins
after 48 hours, which were confirmed by immunoblot analysis, were shown
in photos.

[0053]B) F11 cells were transfected with 0.5 μg of the pcDNA3 vector or
pcDNA3-APP in association with 0.5 μg of the pEFBos vector or the
pEFBos vectors encoding the extracellular domain of the human G-CSF
receptor fused to the full transmembrane domain and a C-terminally
truncated intracytoplasmic domain of gp130 (named "G-CSFR/gp130").
G-CSFR/gp130 (277) contains the full intracytoplasmic domain of gp130
corresponding to amino acids 1-277, while G-CSFR/gp130 (25), (68), and
(133) contain the intracytoplasmic domains corresponding to amino acids
1-25, 1-68, and 1-133, respectively. At 24 hr after transfection, the
cells were added with indicated amounts of human G-CSF. Cell mortality
was determined by Trypan blue exclusion assay at 72 hr after
transfection. The results of immunoblot analysis of the expression of
V6421-APP are shown in photos.

[0054]C) Instead of the pcDNA3-V6421-APP vector, pcDNA3-K595 N/M596L-APP
(NL-APP), M146L-PS1 and N141-I-PS2 vectors were used to do the same
experiments as in FIG. 2A. The results of immunoblot analysis of the
expression of NL-APP, M146L-PS1 and N141-I-PS2 are shown in photos.

[0055]D) F11 cells were transfected with 0.5 μg of the pEFBos vector or
pEF-G85R-SOD1 in association with 0.5 μg of the pEFBos vector or each
pEFBos vector encoding G-CSFR/gp130 (277) or G-CSFR/gp130 (25). At 24 hr
after transfection, the cells were added with indicated amounts of human
G-CSF. Cell mortality was determined by Trypan blue exclusion assay at 72
hr after transfection. The results of the expression of G85R-SOD1, which
were confirmed by the immunoblot analysis, are shown in photos.

[0056]E) Phosphorylation of tyrosine of gp130 was increased by HN
treatment. PCNs (1.0×106 cells/well in 6-well plated) (DIV3)
was transfected with human gp130-encoding adenoviruses. After 60 hrs were
added 1 μM HNG, 1 μM HNA, 100 ng/ml of rat IL-6 and 1 μg/ml of
sIL-6R to the infected cells and incubated at 37 C.° for 15 min.
The results of immunoblot of the precipitate immunoprecipitated with
anti-gp130 antibody with a phosphotyrosine antibody are shown in photos.

[0057]FIGS. 3 (A)-(E)

[0058]A) PCNs, seeded at 2.5 or 5.0×104/well in 96 dishes, were
incubated with or without 25 μM of Aβ (1-43) and in the presence
or the absence of indicated amounts of indicated cytokines. At 72 hr
after incubation, the cells were harvested for WST-8 assays or/and
Calcein fluorescence assays to show cell viability.

[0059]B) The soluble IL-6 receptor-α (sIL-6R) or the soluble CNTF-R
(100 ng/ml) were added to the cells as in the above A), followed by the
stimulation with its ligand, IL-6 or CNTF (100 ng/ml), respectively to
show the effects on the neuronal cell death due to Aβ. The right and
left graphs show the cell viabilities determined by WST-8 assays and
Calcein fluorescence assays, respectively.

[0060]C) The neutralizing antibodies (1 μg) for the mouse gp130, mouse
LIFR, mouse IL-11R and rat CNTF-R were added as in the above A), and cell
viability was determined by Calcein fluorescence assays after 72 hours.

[0061]D) F11 cells were transfected with an indicated amount of
pRNA-U6.1/Shuttle vector (empty), pRNA-U6.1/Shuttle-IL-6R siRNA, or
pRNA-U6.1/Shuttle-LIFR siRNA. At 72 hrs after transfection, total RNA was
extracted, and an amount of IL-6R mRNA and LIFR mRNA was quantatively
measured by real-time PCR. The amount of G3PDH was also determined an
internal control and used for calibration. The change of the proteins was
determined by immunoblot analysis.

[0062]E) F11 cells were transfected with an indicated amount of
pRNA-U6.1/Shuttle vector (empty), pRNA-U6.1/Shuttle-IL-6R siRNA, or
pRNA-U6.1/Shuttle-LIFR siRNA. At 48 hrs after transfection, the cells
were treated with 100 ng/ml IL-6, 100 ng/ml CNTF or 1 μM HNG at
37° for 15 min, and harvested. The results of immunoblot analysis
using anti-phophoSTAT3 (Tyr705) antibody and anti-STAT3 antibody
were shown in photos.

[0063]FIGS. 4 (A)-(C)

[0064]A) F11 cells were transfected with 0.5 μg of the pEF/BOS vector,
pEF-mycHis CREME9, pEF-mycHishuman WSX-1, or pEF-V5-human CNTF-R in
association with 0.5 μg of pCAG-human gp130, or transfected with 0.5
μg of pEF-mycHis human WSX-1 and pEF-V5-human CNTF-R and pCAG-human
gp130. To keep the total amounts of transfected vectors to be 1.5 μg,
an appropriate amount of the pEFBOS vector was added. The graphs show the
results of a binding amount of the biotin-labeled HN to each transfected
cell by the detection of immunofluorescence reaction. The protein
expression by immunoblot analysis is shown below in photos.

[0065]B) F11 cells were transfected with 0.5 μg of pEF-myc His
human CREME9 or pEF-mycHis human WSX-1 in association with 0.5 μg of
pCAG-human gp130. For competition, 10 μM of non-labeled (cold) HNG or
HNA was added for some experiments. Fluorescence signals were detected
with a laser-scanning, confocal microscope LSM (Carl Zeiss, Germany)
(right panels).

[0066]C) The photos show the results of in vitro pull down assay using HN-
or HNA-conjugated Sepharose 4B bead. WSX-1, CNTF-R, and IL-6 were
over-expressed in F11 cells and the pull down assay was made using the
above 4B bead. Immunoblot analysis was done using PO4 antibody against
Humanin (lower panes). HN or HNA comprised in the above Sepharose 4B bead
was compared with synthesized HN peptide (50 pmol) as a positive control.

[0067]FIGS. 5 (A)-(C)

[0068]The graphs show the results obtained by knock-down of the expression
of CNTF-R or WSX-1 in F11 cells by means of plasmid siRNA method, in
order to confirm that these proteins are involved in the HN-mediated
signaling of neuroprotecting function.

[0069]A) F11 cells were transfected with indicated amounts of
pRNA-U6.1/Shuttle vector (NO), pRNA-U6.1/Shuttle-siWSX-1 (W), or
pRNA-U6.1/Shuttle-siCNTF-R (C). Seventy-two hrs after transfection, the
cells were lysed for RNA extraction. The amounts of mRNA were determined
by real-time PCR and the amounts of protein were determined by immunoblot
analysis.

[0070]B) F11 cells were transfected with 0.5 μg of the pcDNA3
vector or pcDNA3-V642I-APP in association with pRNA-U6.1/Shuttle vector
(NO), pRNA-U6.1/Shuttle-siWSX-1 (W), pRNA-U6.1/Shuttle-siCNTF-R (C), or
pRNA-U6.1/Shuttle-siFPR-2 (F) (Hashimoto et al. 2005). At 24 hrs after
transfection, the cells were added with or without 10 nM of HNG. At 72
hrs after transfection, the cells were harvested for WST-8 assays.

[0071]C) F11 cells were transfected with 0.5 μg of the pcDNA3
vector or pcDNA3-V642I-APP in association with pRNA-U6.1/Shuttle vector
(Vec), pRNA-U6.1/Shuttle-siWSX-1 (W), or pRNA-U6.1/Shuttle-siCNTF-R (C)
together with 1 μg of the pEFBos vector, pEF-mycHis-human WSX-1, or
pEF-V5-human CNTF-R. At 24 hrs after transfection, the cells were added
with or without 10 nM of HNG. At 72 hrs after transfection, the cells
were harvested for WST-8 assays.

[0072]FIGS. 6 (A)-(B)

[0073]A) COS7 cells were transfected with 0.5 μg of the pEFBos vector,
pEF-mycHis-human WSX-1, or/and pEF-V5-human CNTF-R. The total amounts of
transfected vectors were 1.0 μg. 72 hrs after transfection, the cells
were harvested for immunoprecipitation with antibodies against myc (for
myc-WSX-1) or anti-hCNTF-R antibody. Resultant precipitates were subject
to immunoblot analysis with a mixture of antibodies against myc and V5.
The results are shown in photos.

[0074]B) F11 cells were treated with 10 μM HNA or 10 nM HNG for 1, 3
and 6 hrs. The cells were treated with 1 mM BS3, as a cross-linker at 30
min before harvesting. The cells were harvested for immunoprecipitation
with antibodies against gp130 or CNTF-R. Resultant precipitates were
subject to immunoblot analysis with antibodies against WSX-1, gp103 and
CNTF-R. A control immunoprecipitation was done using an antibody against
SOD1. Input amounts of lysates were one twentieth the amount used for
immunoprecipitation.

[0075]FIG. 7

[0076]F11 cells were transfected with pcDNA3 vector or pcDNA3-V642I-APP.
At 10 hrs after transfection, the cells were added with 10 nM of HNG, 10
μM of HN or an indicated amount of human IL-27. In some experiments,
the indicated amount of IL-27 or IL-6 was simultaneously administered
with HNG. At 72 hrs after administration, WST-8 assay was carried out.
The expression of V642I-APP was checked as well with respect to the cells
allotted with numbers. The expression of V642I-APP determined by
immunoblot analysis was shown below in a photo.

[0077]FIGS. 8 (A)-(B)

[0078]A) F11 cells were seeded on 96-well plates coated with poly-L-lysine
(7×103 cells/well). An indicated amount of biotin-HN or
biotin-HNG was added to the cells with or without 10 μM of unlabelled
HNG or HNA, followed by a binding assay based on immunofluorescence
reaction (2) (left panels). On the other hand, F11 cells were transfected
with 0.5 μg of pcDNA3.1/GS-human CNTF-R, pEF1/MycHis-human WSX-1, and
1.0 μg of pCAG-human gp130. At 24 hrs after the transfection, the
cells were re-seeded on 96-well plates coated with poly-L-lysine
(7×103 cells/well). After 36 hrs the re-seeding, an indicated
amount of biotin-HN or biotin-HNG was added to the cells with or without
10 μM of unlabeled HNG or HNA, followed by a binding assay based on
immunofluorescence reaction (2) (right panels).

[0079]B) F11 cells were transfected with 0.5 μg of pRNA-U1.6/Shuttle
vector, pRNA-U6.1/Shuttle-WSX-1 siRNA, pRNA-U6.1/Shuttle-CNTF-R siRNA,
both pRNA-U6.1/Shuttle-WSX-1 siRNA and pRNA-U6.1/Shuttle-CNTF-R siRNA
(0.5 μg each), pRNA-U6.1/Shuttle-FPR2 siRNA or pRNA-U6.1/Shuttle-LIFR
siRNA. The total amount of vectors was adjusted to be 1.0 μg by
including a backbone vector. At 24 hrs after the transfection, an
indicated amount of biotin-HNG was added with or without unlabeled HNG,
followed by a binding assay based on immunofluorescence reaction (2) at
72 hrs after the transfection.

[0080]FIGS. 9 (A)-(B)

[0081]A) An indicated amount of biotin-HNG, biotin-HN or human IL-27 was
added to PCN cells that had been treated for three days on 96-well plate
coated with poly-L-lysine (7×104 cells/well), followed by a
binding assay based on immunofluorescence reaction (2). In some
experiments, an indicated amount of unlabeled IL-27, CNTF, IL-6 or HNG
was added simultaneously in addition to 10 nM of biotin-HNG.

[0082]B) An indicated amount of human IL-27, both 10 nM of HNG and an
indicated amount of IL-27, IL-6, CNTF, or 2 μL of an anti-mWSX-1-N
antibody or preimmune sera was added to PCN cells that had been treated
for three days on 96-well plate coated with poly-L-lysine
(5×104 cells/well), followed by the treatment with 10 μM of
Aβ (1-43) at 16 hrs after the addition. WST-8 assay was performed at
72 hrs after the treatment.

[0083]FIGS. 10 (A)-(B)

[0084]A) Immunoblot analysis was performed with the extract of F11 cells
(lane 1). At the same time, immunoprecipitation was performed with the
anti-mWSX-1-C antibody using ten times the amount of said extract (lane
3) or with preimmune serum as a negative control in a
quasi-immunoprecipitation (lane 2).

[0085]B) Immunoblot analysis was performed with the anti-mWSX-1-C antibody
using the extract of PCNs (DIV3) or F11 cells. The results were shown in
photos.

[0086]FIGS. 11 (A)-(B)

[0087]A) F11 cells were added with an indicated amount of HN, HNG or HNA
and harvested 15 min later for immunoblot analysis with antibodies
recognizing phosphotyrosine 706 of STAT3 or STAT3. The results are shown
in photos.

[0088]B) F11 cells were transfected with 0.5 μg of pRNA-U1.6/Shuttle
vector, pRNA-U6.1/Shuttle-WSX-1 siRNA, pRNA-U6.1/Shuttle-CNTF-R siRNA,
both pRNA-U6.1/Shuttle-WSX-1 siRNA and pRNA-U6.1/Shuttle-CNTF-R siRNA
(0.5 μg each), or pRNA-U6.1/Shuttle-FPR2 siRNA. The total amount of
vectors was adjusted to be 1.0 μg by including a backbone vector. At
48 hrs after the transfection, the cells were added with HNG, CNTF or
IL-27 and harvested 15 min later for immunoblot analysis with antibodies
recognizing phosphotyrosine 706 of STAT3 or STAT3. The results are shown
in photos.

[0089]FIGS. 12 (A)-(E)

[0090]A) F11 cells seeded on 6-well plate (7×104 cells/well)
were transfected with 1 μg of pRNA-U6.1/Shuttle vector or
pRNA-U6.1/Shuttle-Bax. At 72 hrs after the transfection, the expression
of mRNA of Bax was determined by real-time PCR. mRNA of G3PDH was
determined as an internal control and used for calibration.

[0091]B) F11 cells seeded on 6-well plate (7×104 cells/well)
were transfected with pRNA-U6.1/Shuttle vector or pRNA-U6.1/Shuttle-Bax
(0.5 μg or 1 μg). At 72 hrs after the transfection, the expression
of Bax protein was determined by immunoblot analysis.

[0092]C) F11 cells seeded on 6-well plate (7×104 cells/well)
were transfected with 1 μg of pRNA-U6.1/Shuttle vector or
pRNA-U6.1/Shuttle-Bax (1 μg). At 72 hrs after the transfection, the
cells were added with 100 nM of Staurosporine (STS) or DMSO. The cell
viability was determined by WST-8 assay after culture for 3, 6 and 9 hrs.
The results obtained with respect to the cells treated with Vector/DMSO
was taken as "100%" and used for calibration.

[0093]D) F11 cells seeded on 6-well plate (7×104 cells/well)
were transfected with 0.5 μg of pcDNA3 vector, pcDNA3-V642I-APP or
pcDNA3-M146L-PS1 in association with 1.0 μg of pRNA-U6.1/Shuttle
vector, pRNA-U6.1/Shuttle-Bax siRNA or pRNA-U6.1/Shuttle-WSX-1 siRNA. At
24 hrs after the transfection, the cells were added with 10 μM of HN,
and subjected to WST-8 assay 72 hrs later. The expression of APP and PS1
was confirmed by immunoblot analysis with respect to parts of the cell
lysate.

[0094]E) F11 cells on 6-well plate (7×104 cells/well) were
transfected with 1.0 μg of pRNA-U6.1/Shuttle vector,
pRNA-U6.1/Shuttle-Bax siRNA, or both pRNA-U6.1/Shuttle-WSX-1 siRNA and
pRNA-U6.1/Shuttle-CNTF-R siRNA (0.5 μg each). At 72 hrs after the
transfection, the cells were added with an indicated amount of biotin-HN
with or without unlabeled HN or HNA (100 μM) and subjected to an
HN-binding assay based on immunofluorescence.

DETAILED DESCRIPTION

[0095]The Humanin receptor or Humanin-like polypeptide receptor (HNR)
according to the present invention comprises at least two kinds of
proteins selected from the group consisting of gp130 or its partial
polypeptide, CNTF receptor a chain (CNTF-R) and WSX-1. Its examples
include the receptor consisting of three proteins, i.e., gp130 or its
partial polypeptide, CNTF-R and WSX-1, the receptor consisting of two
proteins, i.e., gp130 or its partial polypeptide and WSX-1, and the
receptor consisting of two proteins, i.e., CNTF-R and WSX-1. The receptor
may further comprise other proteins as its constituent as long as they
will never deteriorate the function of the receptor of the present
invention.

[0096]Each subunit of the receptor such as gp130 or its partial
polypeptide, CNTF-R and WSX-1 may be modified to have an amino acid
sequence which comprises replacement, deletion, insertion and/or addition
of one or more amino acids as long as such modification will not
deteriorate the function of each subunit. These modified subunits may be
prepared in any method known for those skilled in the art.

[0097]The "Humanin-like polypeptide" comprises a polypeptide and its
derivative, which has suppressing or inhibiting function with a degree of
the same or more than that of the polypeptide of 24 amino acids named
Humanin disclosed in the International Publication No. WO01/021787 for
neuronal cell death caused by AD-related insults. The "Humanin-like
polypeptide (receptor)" in the present specification may also comprise
"Humanin (receptor)" as well.

[0098]One of the examples of Humanin-like polypeptide is therefore a
polypeptide having the amino acid sequence (1) disclosed in the
International Publication No. WO01/021787:

[0099]More particularly, there may be further mentioned polypeptides
disclosed in the International Publication No. WO01/021787, which have an
amino acid sequence selected from the group consisting of SEQ ID NO: 5-8,
10, 12, 13, 21-24, 26-29, 32, 33, 37-40, 46, 48, 54 and 60 wherein one or
more amino acids are replaced, deleted, inserted and/or added, and have a
suppressing or inhibiting function for neuronal cell death caused by
AD-related insults.

[0100]The above polypeptides include various kinds of derivatives. The
"derivatives" means compounds in a modified form wherein their peptide
functional group is modified, added, replaced or deleted by a
conventional way. Such modification of the functional group may be
performed by any known method for the purpose of protection of an
existing functional group, stabilization of polypeptide or controlling of
transition ability into tissues, controlling of polypeptide activity and
the like.

[0101]Thus, the polypeptide may be modified naturally such as by
post-translation modification, or artificially. Modification includes
that of a backbone, an amino acid side chain, a terminal amino acid
group, terminal carboxyl group, group and the like of peptide. The
polypeptide may be a branched- or cyclo- one. The modification includes
acetylation; acylation; ADP-ribosylation; amidation; covalent binding
such as flavin, nucleotide, nucleotide derivative, lipid, lipid
derivative or phosphatidyl inositol; formation of a cross-link;
cyclization; formation of disulfide binding; demethylation;
pyroglutamination; carboxylation; glucosylation; hydroxylation;
iodization; methylation; myristoylation; oxidization; phosphorylation;
ubiquitination and the like. Furthermore, the above polypeptide may be in
a form of its salt or ester. The polypeptide may be synthesized according
to a known synthetic technique, or prepared by expression of a DNA
encoding said polypeptide.

[0102]The phrase "have a suppressing or inhibiting function for neuronal
cell death caused by AD-related insults" in the present specification
means being able to suppress or inhibit at least one kind of neuronal
cell death related to AD. Thus, the above Humanin-like polypeptide
includes a polypeptide that has a function of inhibiting at least one
kind of neuronal cell death related to AD. The neuronal cell death may
not be necessarily completely inhibited, but be significantly inhibited.
The neuronal cell death may be determined by the method described in the
following Example or by other methods such as that disclosed in the
International Publication No. WO00/14204.

[0103]A compound that binds to the Humanin-like polypeptide receptor may
be identified by the method for screening according to the present
invention. The compound may be originally comprised in a living body such
as human, or artificially synthesized. The compound may bind to any part
of the Humanin-like polypeptide receptor, such as its intracellular
domain or extracellular domain. The compound may be an agonist or
antagonist for the receptor.

[0104]The screening method according to the present invention may be
carried out in any known method or system such as a cell system or a
cell-free system. The cell system uses cells per se that express the
Humanin-like polypeptide receptor. As the proteins constituting the
Humanin-like polypeptide receptor have been first revealed by the present
invention, the cell in which the Humanin-like polypeptide receptor is
compulsorily (constitutively) expressed may be prepared by any method
known for those skilled in the art. For example, such cell may be easily
obtained by transformation of an appropriate host cell with an expression
vector comprising a gene encoding at least one of the proteins
constituting the Humanin-like polypeptide receptor. By using such
transformed cell, the binding characteristics between a compound
comprised in a subject sample and the Humanin-like polypeptide receptor
may be increased. As a result, even if only a small amount of a target
compound is comprised in the subject sample, or if a binding capacity
(affinity) of the compound is relatively small, its binding
characteristics may be significantly determined.

[0105]The screening method of the present invention may be carried out by
the following steps: [0106](a) a step of placing a subject sample in
contact with Humanin receptor or Humanin-like polypeptide receptor (HNR)
or at least one protein that constitutes it; [0107](b) a step of
determining a binding characteristics between the receptor and the
compound comprised in the subject sample; and [0108](c) a step of
selecting the compound that binds to the receptor.

[0109]By performing the step (a) in the presence of Humanin or
Humanin-like polypeptide the binding characteristics of the compound may
be determined by means of a competitive reaction between the compound and
the Humanin or Humanin-like polypeptide.

[0110]In the screening method carried out in the cell system, the contact
between the subject sample and the receptor may be realized by any method
known for skilled in the art, such as adding the subject sample into a
culture system of the cell expressing the receptor. In such cell system,
the binding characteristics between the receptor and the compound is
determined by detecting a change (increase, decrease or inhibition) in
suppressing or inhibiting function for the neuronal cell death.
Furthermore, the binding characteristics between the receptor and the
compound may be determined by detecting increase or decrease of
phosphorylation of tyrosine at 706 of STAT3.

[0111]The expression vector may be easily prepared by any method known for
those skilled in the art. The gene encoding at least one of the proteins
constituting the Humanin-like polypeptide may be easily prepared based on
the disclosures of the International Publication No. WO01/021787 pamphlet
and other known publications. The expression vector may comprise 5' and
3' non-coding regions such as, for example, a non-transcription sequence,
non-translation sequence, promoter, enhancer, suppressor, transcription
factor-binding sequence, splicing sequence, poly A--adding sequence,
IRES, mRNA-stabilizing or destabilizing sequence in addition to a coding
region of the protein.

[0112]There is no limitation on a kind of the host cell used in the
screening method of the present invention, including cells or its bodies
of mammalian such as human and monkey, plants, and insects. A host-vector
system includes baculovirus-Sf cell system (Okamoto et al., J. Biol.
Chem. 270:4205-4208, 1995), pcDNA-CHO cell system (Takahashi et al., J.
Biol. Chem. 270:19041-19045, 1995), and CMV promoter-plasmid-COS sell
system (Yamatsuji et al., EMBO J. 15:498-509, 1996). These cells may be
cultured by any method known for those skilled in the art.

[0113]It is not necessary for such host cells originally express the HNR
by themselves. However, the host cells may be prepared from tissues or
cells that are supposed to express the receptor, such as brain cortex
tissue, neuronal cell strains, neuroblastoma or teratoma. The neuronal
cells include F11 cells, PC12 cells (L. A. Greene and A. S. Tischler,
1976, Proc. Natl. Acad. Sci. USA 73:2424-2428), NTERA2 cells (J.
Skowronski and M. F. Singer, 1985, Proc. Natl. Acad. Sci. USA
82:6050-6054), and SH-SY5 Y cells (L. Odelstad et al., 1981, Brain Res.,
224:69-82). In such cases, the compulsory expression of the receptor from
the introduced expression vector would produce more amount of the HNR
than originally expressed in these cells, promoting sensitivity of the
detection well.

[0114]The screening method according to the present invention may be also
carried out in the cell-free system by any method known for those skilled
in the art. For example, the receptor or one of its constituting proteins
may be used in its soluble form or a form immobilized or bound to a
carrier depending on the screening method. The receptor of the present
invention may be labeled with, for example, a radioactive isotope,
fluorescent substance, biotin or digoxgenin, a tag sequence.

[0115]For example, the screening method may be carried out by placing the
subject sample on an affinity column containing the HNR or one of its
constituting proteins immobilized thereto, and purifying a compound that
specifically binds to the column. Alternatively, the same method may be
carried out by reacting a synthetic compound, natural product bank, or
random phage peptide display library with the immobilized HNR or one of
its constituting proteins. The screening may be made by using surface
Plasmon resonance phenomenon (for example, manufactured by BIAcore Co.).
These screening method may be carried out as a high through-put system be
means of combinatory chemistry technique.

[0116]The subject sample to be used in the screening method according to
the present invention includes a purified protein such as an antibody, an
expressed product from a gene library, cell extract, supernatant obtained
from cell culture, library of synthetic low-molecular compounds, natural
materials such as soil, and cell-producing substances such as broth of
actinomycetes. The subject sample may be optionally labeled with a
radioactive isotope, fluorescent substance, etc.

[0117]Those skilled in the art may easily prepare a cell in which a gene
encoding at least one protein that constitutes Humanin receptor or
Humanin-like polypeptide receptor (HNR), which is selected from the group
consisting of gp130, CNTF-R and WSX-1, is knocked out, by conventional
gene-targeting technique. Such knockout cell is preferably a mammalian
cell such as mouse and human cells. A knockout animal may be further
generated by using these knockout cells according to any method known for
those skilled in the art. The knockout animal may be heterozygous or
homozygous. Especially, knockout rodents such as mouse or rat are useful
as an experimental animal for researches of neurodegenerative diseases
such as AD.

[0118]Since the compound that binds to Humanin receptor or Humanin-like
polypeptide receptor (HNR) has an agonist or antagonist activity for the
Humanin, it may be used in prevention or treatment of neurodegenerative
diseases such as Alzheimer's disease (AD), amyotrophic lateral sclerosis
(ALS), mad cow disease, vascular dementia (VD).

[0119]As prior studies have revealed that neuronal cell death occurs in
AD, the pharmaceutical composition according to the present invention is
expected be used as a medicine for protection of neurodegeration in AD.
The pharmaceutical composition according to the present invention may be
also used to prevent or treat diseases caused by neuronal cell death due
to brain ischemia (T. Kirino, 1982, Brain Res., 239:57-69),
Parkinsonism-dementia complex (PDC) (M. H. Polymeropoulos et al., 1997,
Science, 276:2045-2047), Lewy bodies (M. G. Spillantini et al., 1998,
Proc. Natl. Acad. Sci. USA, 95:6469-6473), and Down syndrome-related
dementia. As APLP1, an analogous molecule of APP is thought to be a
causative gene of congenital nephrosis syndrome (Lenkkeri, U. et al.,
1998, Hum. Genet. 102:192-196), renal diseases such as nephrosis syndrome
may be a target of prevention or treatment by the pharmaceutical
composition according to the present invention.

[0120]The pharmaceutical composition according to the present invention
comprises the compound that can bind to the HNR as an effective component
may be directly administered into a patient, or formulated by known
formulation methods optionally with, for example, a pharmaceutical
carrier or solvent such as sterilized water, physiological saline,
vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, and
slow-releasing agent. The pharmaceutical composition according to the
present invention may be in a form of aqueous solution, tablet, capsule,
troche, buccal tablet, elixir, suspension, syrup, nasal solution, or
inhalant liquid. The content of the effective component may be optionally
determined by those skilled in the art, depending on the purpose of the
treatment, administration route, subject to be treated and the like.

[0121]The pharmaceutical composition according to the present invention
may be administered transdermally, transnasally, transbronchially,
intramuscularly, intraperitoneally, intravenously, through spinal foramen
or cerebral ventricle or orally, depending on the features of the
component. In the treatment of cerebral neurodegenerative diseases, the
pharmaceutical composition according to the present invention may be
preferably introduced into central nerve system through an appropriate
route such as an intravenous, through spinal foramen or cerebral
ventricle and intradural injection. Those skilled in the art may select
an appropriate dose depending on the age, weight and conditions of
disease of a patient and the administration route and the like. The dose
and the administration route may be in turn optionally selected by those
skilled in the art depending on tissue-transition ability of the
effective component, the purpose of treatment, the age, weight and
conditions of disease of a patient and the like. In the administration of
the pharmaceutical composition according to the present invention for the
purpose of protection of cerebral neurodegeneration such as in AD
disease, the composition is preferably administered in such an amount as
to effectively inhibit neurodegeneration around target cells. Thus,
Humanin polypeptide or other substances showing an equivalent protecting
function for neuronal cell death may be administered in an amount of at
least 1 nM, preferably 10 nM or more, more preferably 100 nM or more,
further preferably 1 μM or more.

[0122]The antibody according to the present invention may be in any forms
or kinds known for those skilled in the art, including polyclonal
antibodies and monoclonal antibodies, and various kinds of chimeric
antibodies such as a humanized one, which may be prepared by any genetic
engineering method known for those skilled in the art.

EXAMPLES

[0123]The present invention will be further explained more in detail by
referring to the following examples, which should not be construed to
limit a technical scope of the invention.

[0134]The transfection procedures were as described (Hashimoto et al,
2000, 2001a, 2003). F11 cells, seeded at 7×104/well in 6-well
dishes, were transfected with indicated vectors. Transfection efficiency
in these protocols has been determined to be invariably around 70%. At 72
hrs after transfection, the Trypan blue exclusion assay and LDH assay
were performed as a cell-death assay and the WST-8 assay was performed as
cell-viability assays (Hashimoto et al, 2000, 2001a b, 2003). HN was
added to the culture medium usually at 5 hrs after transfection, but in
some cases at 24 hrs after transfection.

Primary Cortical Neurons And Cell Viability Assays

[0135]The primary culture of mouse cortical neurons was prepared as
described previously (Sudo et al., 2000). Briefly, primary cortical
neurons, obtained from embryonic day 14 (E14) ICR mice were seeded in
poly-L-lysine-coated 96 well plates (Sumitomo Bakelite) at 2.5 or
5.0×104 cells/well in Neuron Medium (Sumitomo Bakelite)
(Hashimoto et al., 2003; Niikura et al., 2004). Purity of neurons by this
method was 22 98%. After 3 days, the culture medium was replaced with
DMEM with N2 supplement. On the fourth day in vitro, 25 μM of Aβ
(1-42) was added in association with indicated concentrations of HN or
cytokines in the presence or the absence of soluble cytokine receptors or
neutralizing antibodies. At 72 hrs after the onset of treatment, cell
viability was assessed by WST-8 assay and/or Calcein fluorescence assay
and cell mortality was assessed by the Trypan blue exclusion assay and
LDH assay (Hashimoto et al, 2000, 2001 a b, 2003).

Immunofluorescence-Based Binding Assay (1)

[0136]F11 cells (7×104/well in 6-well plates) were replated
into 96-well plates (7×103 cells/well) at 24 hrs after
transfection with indicated amounts of the plasmids encoding mycHis-WSX
or V5-CNTF-R, and with PCAG-human gp130 if required. At 36 hrs after
transfection, the cells were added with 100 nM of biotin-labeled HNG-FLAG
in the presence or the absence of 10 μM of HNG (S14 G-HN) or HNA (C8
A-HN) (Hashimoto et al., 2001a). After 6 hrs incubation, they were fixed
with 4% paraformaldehyde in PBS for 30 min. After washing with PBS, cells
were stained with FITC-conjugated avidin (Molecularprobe, Eugene, Oreg.,
USA). Immunofluorescence intensity was measured (excitation=485 nm,
emission=535 nm) with a spectrofluorometer (Wallac1420 ARVOsx Multi Label
Counter). Immunohistochemical analysis was done with a laser-scanning,
confocal microscope LSM (Carl Zeiss, Germany).

Immunofluorescence-Based Binding Assay (2)

[0137]F11 cells (7×104/well in 6-well plates) were replated
into 96-well plates (7×103 cells/well) at 24 hrs after
transfection with indicated amounts of the plasmids encoding mycHis-WSX
or V5-CNTF-R, and with PCAG-human gp130 if required. At 36 hrs after
transfection, the cells were added with biotin-labeled HN or HNG of
indicated concentrations in the presence or the absence of HNG
(S14G-HN)or HNA (C8A-HN) of indicated concentrations (Hashimoto et al.,
2001 a). After 6 hrs incubation, they were fixed with 4% paraformaldehyde
in PBS for 30 min. After washing with PBS, cells were stained with
FITC-conjugated avidin (Molecularprobe, Eugene, Oreg., USA).
Immunofluorescence intensity was measured (excitation=485 nm,
emission=535 nm) with a spectrofluorometer (Wallac1420 ARVOsx Multi Label
Counter). Immunohistochemical analysis was done with a laser-scanning,
confocal microscope LSM (Carl Zeiss, Germany).

Pull-Down Assays

[0138]F11 cells (7×104/well in 6-well plates) were transfected
with the plasmids encoding mycHis-WSX-1, V5-CNTF-R or rat IL-6R (V6
tagged). At 48 hrs after transfection, the cells were harvested for
pull-down assays with HN or HNA-conjugated Sepharose 4B. For conjugation
of HA or HNA with Sepharose 4B, 3 ml of CNBr-activated Sepharose 4B was
incubated with 0.5 mg of HN or HNA in a coupling buffer (0.1M NaHCO3
0.5M NaCl, pH8.3) overnight at 4° C. The beads were then reacted
with a blocking buffer (0.2M glycine, pH 8.0) for 2 hrs at a room
temperature, washed with the coupling buffer and stored at 4° C.
for use in pull-down assay. For each pull-down assay, 20 μl of 1:1
slurry of Sepharose was used for 100 μl of the cell lysate.

Immunoblot Analysis

[0139]Cell lysates (10-20 μg/lane) or pulled-down precipitates were
subject to SDS-PAGE, and the proteins separated on the gel were
transferred onto polyvinylidene difluoride membranes as described
(Hashimoto et al, 2000). Visualization of the immunoreactive protein
bands was performed by ECL (Amersham Pharmacia Biotech, Uppsala, Sweden).

Plasmid-Based Small Interfering RNA

[0140]Plasmid vectors encoding small interfering RNA (siRNA) for mouse
FPR2, mouse CNTF-R, mouse WSX-1, mouse IL-6R and mouse LIFR were
constructed as follows. The sense and antisense DNA fragments used for
the construction were as follows:

[0141]These DNA fragments were annealed by heating and cooling according
to the manufacturer's instruction. These annealed primers and the pRNA
U6.1/Shuttle empty vector (GenScript, NJ, USA) were digested by BamHI and
KpnI at 37° C. overnight. The digested DNA fragments and the empty
vectors were purified by GENE CLEAN II kit (Q BIOgene, USA). Ligation was
performed with Ligation Convenience Kit (NIPPON GENE, Tokyo, Japan)
according to the manufacturer's instruction. The sequence of these siRNA
vectors was confirmed by a direct sequencing, and effects of these siRNA
plasmids were confirmed by real-time PCR as described below.

Real-Time PCR

[0142]We performed real-time PCR to assess the amount of endogenous mRNA.
Cells were harvested for RNA extraction with ISOGEN reagent (Nippon Gene,
Toyama, Japan) followed by real-time PCR. The first-strand cDNAs were
synthesized using Sensiscript reverse transcriptase (QIAGEN, Germany)
with 0.5 mg total RNA. Real-time PCR analysis was performed using a
QuantiTect SYBR Green PCR kit (QIAGEN), followed by analysis with ABI
PRISM7700 (Applied Biosystems, Foster City, Calif.). We made sets of a
sense primer and an antisense primer as follows:

[0143]Data analysis was performed using a software Sequence Detection
System ver. 1.9.1 (Applied Biosystems). To adjust the expression level of
each mRNA, G3PDH mRNA was used as an internal control.

Statistical Analyses

[0144]All cell-death (mortality) experiments, cell viability experiments,
and real-time PCR experiments were done with n=3. All values in the
figures of the in vitro study are mean±SD. Statistical analysis was
performed with analysis of variance followed by post hoc test, in which
<0.05 was assessed as significant.

Result 1

[0145]Considering that certain tyrosine kinases as well as STAT3 are
involved in HN-mediated neuroprotection (Hashimoto et al. 2005), we
suspect that the HN receptor belongs to a cytokine receptor family. Gp130
is a cytokine receptor subunit common to the cytokine receptors belonging
to the IL-6 receptor family. As shown in FIGS. 1A and 1B, enforced
expression of the extracellular domain and the transmembrane domain of
human gp130 (gp130 tr) or addition of recombinant soluble human gp130
consisting of the extracellular domain of human gp130 (gp130ED), resulted
in complete suppression of HN-mediated neuroprotection against toxicity
by overexpressed V642I-APP(A) and 25 μM of Aβ (B). Because gp130
tr and gp130ED has been demonstrated to act a dominant-negative form of
gp130 (Kumanogoh et al., 1997; Jostock et al. 1998), this finding
indicated that HN-mediated neuroprotective signal is mediated by gp130.
We then tested how treatment with neutralizing anti-gp130 antibody would
modify HN-mediated signals in order to confirm this finding. Antibodies
to mouse gp130 (RX435), which has been shown to inhibit mouse gp130
function, but not human gp130, attenuated HN-mediated neuroprotection
(FIG. 1C) while such inhibition was suppressed by simultaneous ectopic
expression of human gp 130 in F11 cells (FIG. 1D), clearly indicating
that gp 130 is involved in HN-mediated signals.

Result 2

[0146]To further confirm the involvement of gp130 in HN-mediated
neuroprotective signals, we constructed various chimera proteins
consisting of the extracellular domain of the G-CSF receptor fused to the
full transmembrane domain of gp130 and various-length intracellular
domains of gp130 systematically C-terminally truncated (Fukuda et al.,
1996). A chimera protein named G-277 contained the 277 amino acid-long
full intracellular domain of gp130 while G-195, G-133, G-68, and G-25
contained the amino acid 1-195, 1-133, 1-68, and 1-25 intracellular
portions, respectively (The N-terminal amino acid in the intracellular
domain is considered as No. 1). At first, the expression of these chimera
proteins was confirmed (FIG. 2A). Stimulation with 100 nM G-CSF prevented
neuronal cell death induced by ectopic expression of V642-APP when G-277
was expressed. On the other hand, the same stimulation did not prevent
V642I-APP mediated neuronal cell death when either G-25 or G-68 was
expressed (FIG. 2B). Intermediately, stimulation with 100 nM G-CSF
partially prevented V642I-APP-mediated neuronal cell death when G-133 was
expressed (FIG. 2B). Because it was already shown in the previous study
that the third tyrosine from the membrane of the intracellular domain of
gp130, which is contained in G-133, was essential for anti-apoptotic
effect in proB cells (Fukuda et al., 1996), we speculated that other
signal mediated by the 134-277 (amino acids)-corresponding part of the
intracellular domain of gp130 was required for the full protection of
neuronal cell death by AD-related insults. Similarly, stimulation with
100 nM G-CSF prevented NL-APP-, M146L-PS1-, N141I-PS2-mediated neuronal
cell death when G-277 was expressed, but not when G-25 was expressed
(FIG. 2C). In contrast to these AD-related neuronal cell death,
stimulation with 100 nM G-CSF did not prevent neuronal cell death induced
by overexpression of the mutant Cu/Zn-superoxide dismutase genes, which
has been shown to cause familial amyotrophic lateral sclerosis, a
representative motoneuron-specific neurodegenerative disease (FIG. 2D).
Furthermore, the treatment with HN increased phosphorylation level of
gp130 (FIG. 2E).

Result 3

[0147]In order to search a molecular basis of the HN receptor, we tested
whether or not known cytokines belonging to IL-6 families could mimic the
HN-mediated neuroprotection. As shown in FIG. 3A, treatment with either
physiological levels (up to 100 ng/ml) of mouse cardiotropin-1 (CT-1),
rat IL-6, mouse IL-11, human OncostatinM (OSM), mouse Leukemia-inhibitory
factor (LIF), or human Ciliary Neurotrophic Factor (CNTF) that could bind
and stimulate mouse cognate receptors, did not inhibit neurotoxicity by
Aβ in F11 cells. This was true for neurotoxicity by overexpression
of V642I-APP (similar data not shown).

[0148]Because IL-6 receptor, IL-11 receptor, the LIF receptor, the CNTF
receptor, and gp130 were expressed in F11 cells as well as primary
cortical neurons (PCNs) (unpublished observation by Y. H. and M. M.), the
functional IL-6, IL-11, OS, LIF, and CNTF-R must have been generated in
these neuronal cells by combination of these receptor subunits.
Accordingly, we concluded that IL-6-, IL-11-, CT-1-, OSM-, LIF-, and
CNTF-induced activation of gp130-mediated pathways was insufficient for
protection against neuronal cell death by AD-related insults. These
findings were against the possibility that HN would elicit
neuroprotection by binding to these receptors.

Result 4

[0149]To increase IL-6-mediated signal, we examined the effect of addition
of the soluble IL-6 receptor a or the soluble CNTF receptor a (sIL-6R)
(100 ng/ml) in association with treatment with IL-6 or CNTF (100 ng/ml)
on neuronal cell death by AP (FIG. 3B). Either IL-6 completely mimicked
HN in neuroprotection in the presence of overexpression of sIL-6R,
indicating that enhancement of IL-6-induced homodimerization of gp130
mimics the HN activity. In contrast, CNTF did not mimic HN even in the
presence of overexpression of the soluble CNTF receptor. Considering that
CNTF binding to the CNTF receptor induced heterodimerization between
gp130 and the LIF receptor so as to trigger the intracellular signal
cascade, we speculated that the LIF receptor was not involved in
HN-mediated neuroprotection.

Result 5

[0150]Using neutralizing antibodies to gp130-coupled receptors, we further
examined whether or not known gp130-coupled receptors participated in
HN-mediated neuroprotection. We then found that a neutralizing antibody
to the rat CNTF receptor, which was speculated to also recognize mouse
CNTF receptor, was able to nullify the HN activity (FIG. 3C). Combined
with the foregoing finding about the CNTF/soluble CNTF receptor in FIG.
3B, we suspected that the CNTF receptor was involved in HN signals in a
manner quite different from the way in which association between CNTF and
CNTF-R facilitated the heterodimerization of gp130 and the LIF receptor.
We further constructed vectors encoding siRNA specific to IL-6R and
LIF-R, respectively (FIG. 3D). The decrease of expression of endogenous
IL-6R or LIF-R due to the expression of these vectors in F11 cells did
not attenuate phosphorylation of STAT3 (FIG. 3E), showing that HN did not
transduce cell viability signal through IL-6R or LIF-R.

Result 6

[0151]In search for molecular basis of the HN receptor as a putative
gp130-coupled receptor complex, we have further tested the recently
identified IL-27 receptor WSX-1 (Specher et al., 1998), which appear to
be a gp130-coupled receptor (Planz et al., 2004), and an uncharacterized
putative gp130-coupled receptor, CREME9 (CRL4) (Boulay et al., 2003) by
an immunofluorescence-based HN binding assay (1) after ectopic
overexpression of these genes together with human gp130 in F11 cells.
This assay was not sensitive enough to detect association between HN and
the endogenous HN receptor.

[0152]We found that expression of human WSX-1 or the human CNTF receptor
in F11 cells increased the binding of HN to the F11 cells while
expression of CREME9 did not (FIG. 4A). We further found that
overexpression of both the CNTF receptor and WSX-1 resulted in
synergistic increase in binding of HN to the F11 cells (FIG. 4A),
suggesting that both the CNTF receptor and WSX-1 were components of the
HN receptor. The expression of each protein was confirmed by immunoblot
analysis. To confirm that HN specifically associated with WSX, we
performed an HN-binding experiment in the presence of a large amount of
HN-G, a 1000-fold potent HN derivative, or HN-A, an HN mutant with null
activity as a negative control (Hashimoto et al., 2001 a) (FIG. 4B).
Apparently, HN-G, but not HN-A, nullified the binding of HN to cells
expressing WSX-1 and gp130, confirming the presence of a specific binding
between HN and WSX-1. Using the in vitro pull-down assays with HN (or
HN-A) covalently immobilized onto Sepharose 4B beads, we further
confirmed that HN bound to the CNTF receptor and WSX-1, but not to the
IL-6 receptor in the lysates prepared from F11 cells overexpressing
CNTF-R or WSX-1 (FIG. 4C). On the other hand, it was confirmed that HNA
did not bind to these receptors (FIG. 4C).

Result 7

[0153]To confirm the involvement of the CNTF receptor and WSX-1 in the
HN-mediated neuroprotective signal, we knocked down expression of these
proteins by using a plasmid-based siRNA-mediated disruption technique.
The efficacy of this method was confirmed by measurement of mRNA with
real-time PCR (Sui et al., 2001; Kanekura et al., 2004) (FIG. 5A). As a
negative control, we tested siRNA for mouse FPR-2 (Hashimoto et al.,
2005), a putative HN receptor (Ying et al., 2004). As shown in FIG. 5B,
disruption of the endogenous WSX-1 almost completely suppressed the HN
activity against neurotoxicity by overexpression of V642I-APP. Disruption
of the CNTF receptor reduced the HN activity by 30% compared with the
control while disruption of FPR-2 did not reduce the HN activity.
Furthermore, coexpression of human CNTF receptor or human WSX-1
completely recovered the HN activity, which had been inhibited by siRNA
for the mouse CNTF receptor or mouse WSX-1 (FIG. 5C), strongly supporting
the notion that the CNTF receptor and WSX-1 were components of the HN
receptor. Considering that treatment with the neutralizing antibody to
the CNTF receptor completely antagonized the HN activity (FIG. 3C) while
siRNA-mediated disruption of the CNTF receptor appeared incomplete
compared with that of WSX-1 (FIG. 5A), we suspected that incomplete
disruption of expression of the CNTF receptor could result in incomplete
inhibition of the HN activity in FIG. 5B.

Result 8

[0154]We then tested whether or not CNTF-R could make a complex with
WSX-1. To address this issue, we overexpressed myc-tagged human WSX-1 and
V5-tagged human CTNFR in COST cells and performed co-immunoprecipitation
analysis. As shown in FIG. 6A, immunoprecipitation of WSX-1
co-precipitated CNTF-R and immunoprecipitation of CNTF-R co-precipitated
WSX-1, indicating that WSX-1 could associate with CNTF-R.

Result 9

[0155]The HN treatment induced the association between CNTF-R and WSX-1,
or between WSX-1 and gp130. As shown in FIG. 6B, F11 cells were harvested
at 0, 1, 3 and 6 hrs after the treatment with 10 nM of HNG or HNA and
subjected to immunoprecipitation analysis with the anti-gp130 antibody or
anti-CNTF-R antibody. The resulting precipitates were then subjected to
immunoblot analysis with the anti-mWSX-1 antibody. The results showed
that the HN treatment would specifically induce the association between
CNTF-R and WSX-1 and between WSX-1 and gp130 at an endogenous expression
level.

Result 10

[0156]It had been already known that WSX-1/gp130 functioned as a receptor
of IL-27, indicating the possibility that IL-27 could show the same
effect as HN. As expected, the IL-27 treatment showed HN-like effect
(activity) at a higher concentration range (1-10 μM). It was also
found that the IL-27 treatment suppressed the HN effect at a range of 100
nM or less (FIG. 7).

Result 11

[0157]We have developed a highly-sensitive immunofluorescence-based
binding assay (2) and succeeded in detecting the association between HN
and the endogenous HN receptor. This binding assay was performed using
F11 cells comprising HNR at an endogenous level (FIG. 8A, left two
panels), and F11 cells transfected with CNTF-R/WSX-1/gp130 so as to
highly express them (right two panels). The upper two panels show the
binding of HN, and the lower two panels show the binding of HNG. These
results indicated that HN and HNG showed concentration-dependent binding
with saturation at 10 μM and 10 nM, respectively, each KD being in a
range of between 1-10 μM and 1-10 nM, respectively. The specificity of
their binding was confirmed by the fact that over-addition of unlabeled
HN and HNG could almost completely inhibit the binding. Binding
parameters between HN or HNG and HNR that were obtained from the above
results almost completely coincided with their inhibiting parameters of
neuronal cell death already reported (Hashimoto et al., 2001 a, b) (FIG.
8A).

Result 12

[0158]We confirmed that the binding between HN or HNG and HNR at the
endogenous level would depend on the expression of CNTF-R and WSX-1 by
verifying that the binding was affected by the increase of the expression
of CNTF-R and WSX-1 in F11 cells by means of siRNA method (FIG. 8B).
Thus, as shown in FIG. 8B, the binding was significantly decreased
between HN or HNG, and F11 cells in which the expression of the
endogenous CNTF-R or WSX-1 was decreased, and the above binding was
almost completely inhibited in the F11 cells in which the expression of
the endogenous CNTF-R and WSX-1 was simultaneously decreased. The binding
was not be affected by the decrease of the expression of FPR2 or LIFR.
The above results indicate that the binding between HN or HNG and F11
cells depends on the expression of CNTF-R and WSX-1.

Result 13

[0159]We further studied whether or not the treatment of PCN, a more
physiological neuronal cell, with a high concentration of IL-27 and CNTF
would inhibit the binding between PCN and HN in a similar manner as with
F11 cells. Like the results with F11 cells, it was revealed that the
IL-27 treatment in a range of 100 μM-1 μM inhibited the binding of
HN to PCN (FIG. 9A). It was also observed that CNTF-R would inhibit the
binding of HN to PCN in a similar range, but IL-6 did not show such
effect.

Result 14

[0160]In accordance with the above binding-inhibition tests, it was shown
that IL-27 had the HN-like effect at a higher concentration range while
it suppressed the HN effect at a lower concentration also with respect to
PCN in a similar pattern as in F1 cells (FIG. 9B). It was further
observed that CNTF inhibited the HN effect like IL-27, and that the
HN-effect was also inhibited by the treatment with the anti-WSX-1
antibody (mWSX-1-N). These results supported that CNTF-R/WSX-1/gp130
would function as a receptor of HN even in PCN just like in F11 cells.

Result 15

[0161]Actually, the expression of WSX-1 was confirmed also in PCN like in
F11 cells (FIGS. 10A and 10B).

Result 16

[0162]It was observed that HN and HNG increased phosphotylation of
tyrosine 706 of STAT3 in F11 cells like the treatment with CNTF and IL-27
(FIG. 11A). Furthermore, the decrease of the expression of the endogenous
CNTF-R or WSX-1 almost completely inhibited the phosphotylation of
tyrosine 706 of STAT3 (FIG. 11B). These results indicated that HN or HNG
induced phosphotylation of tyrosine 706 of STAT3 dependently on CNTF-R or
WSX-1.

Result 17

[0163]An siRNA specific to mouse Bax (siRNA-Bax) was prepared. The
expression of siRNA-Bax in F11 cells significantly decreased an amount of
endogenous Bax mRNA and its protein (FIGS. 12A, B). The siRNA-Bax also
showed a significant inhibiting effect for apoptosis of F11 cells induced
by Staurosporine (STS), leading to the conclusion that siRNA-Bax did work
effectively (FIG. 12 C). Then, we tested whether or not the inhibition of
the expression of endogenous Bax in F11 cells by siRNA-Bax could change
the inhibiting effect of HN for F11 neuronal cell death induced by a high
expression of V642I-APP or M146L-PS1. The effect of siRNA for WSX-1 was
first examined as a positive control. However, since it was revealed that
the siRNA-Bax had the inhibiting effect for the neuronal cell death due
to V642I-APP by itself, the effect of siRNA-Bax for the HN activity could
not be verified in a system detecting the neuronal cell death due to
V642I-APP. On the other hand, as the siRNA-Bax did not have the
inhibiting effect for the neuronal cell death due to M146L-PS1, the
effect of siRNA-Bax for the HN activity could be detected. As shown in
FIG. 12D, the inhibition effect of HN for the neuronal cell death induced
by M146L-PS1 was not affected by the inhibition of the expression of the
endogenous Bax at all. It was therefore concluded that the inhibition
effect of HN for the neuronal cell death did not exert via the inhibition
of the function of Bax. Furthermore, FIG. 12 E showed that the inhibition
of the expression of the endogenous Bax did not suppress the binding
activity of HN to F11 cells at all, unlike the inhibition of the
expression of endogenous WSX-1 and CNTF-R. These two results showed that
an intracellular Bax was not a target for HN at least in F11 cells.

[0164]The contents of the scientific journals listed below are cited and
incorporated into the present specification as a part of its disclosure.

[0230]Humanin-like polypeptide receptor (HNR) revealed by the present
invention is useful in revealing a mechanism of promoting or suppressing
the intracellular signal transduction for showing neuroprotecting
activity of HN, and is utilized in a clinical application of development
of a drug for the treatment of neurodegenerative disease, especially
Alzheimer's disease.